1. Field of the Invention
This invention relates to the field of DNA amplification and analysis. More particularly, this invention relates to a system and method for releasing DNA from cells, for amplifying the DNA, and for detecting the amplified DNA products, wherein the device is formed from multiple layers of green-sheet that have been sintered together to form a substantially monolithic structure.
2. Description of Related Art
The conventional way of analyzing the DNA present in a sample of cells involves performing multiple steps using several different bench top instruments in a laboratory setting. First, the DNA must be extracted from the cells in the sample. This is typically done by performing any number of cell lysing procedures that cause the cells to break apart and release their contents. Next, the DNA is typically separated from the rest of the cell contents, as the presence of other cell contents may be undesirable in subsequent steps. To obtain an amount of DNA suitable for characterization, the DNA is amplified, such as by using the polymerase chain reaction (PCR). The resulting amplified DNA products can then be identified by any number of techniques.
The ability to perform all of these steps in a single miniaturized device has the potential for saving time and expense. Such miniaturized devices can be made much more portable than conventional apparatus, thereby enabling samples to be analyzed outside of the laboratory, such as the location where the samples are collected. A miniaturized DNA analysis device can also allow the analysis steps to be automated more easily. As a result, assays could be performed by less highly trained personnel than presently required.
Most efforts at fabricating miniaturized DNA analysis devices have focused on silicon as a substrate. For example a microchip device made out of silicon that performs the steps of cell lysis, PCR amplification, and electrophoretic analysis has been reported. See Larry C. Water, et al., xe2x80x9cMicrochip Device for Cell Lysis, Multiplex PCR Amplification, and Electrophoretic Sizing,xe2x80x9d Anal. Chem., 70:158-162 (1998). Similarly, U.S. Pat. Nos. 5,639,423, 5,646,039, and 5,674,742 each disclose a microfabricated silicon device suited for performing PCR.
Silicon, however, suffers from a number of disadvantages as a substrate material. The cost of fabricating microfluidic devices in silicon can be relatively high. Silicon""s high thermal conductivity can make the thermal cycling needed to perform PCR difficult, and silicon""s property of being electrically semiconducting can hamper the operation of components that require the maintenance of a high potential difference. Most importantly, however, the difficulty of bonding multiple layers of silicon together makes it difficult to integrate complex components into the device.
In a first principal aspect, the present invention provides a multilayered microfluidic DNA amplification device comprising a substantially monolithic structure formed from a plurality of green-sheet layers sintered together. The green-sheet layers include particles selected from the group consisting of ceramic particles, glass particles, and glass-ceramic particles. The substantially monolithic structure has a fluid passageway defined, wherein the fluid passageway includes an inlet port for receiving fluid and a DNA amplification chamber for amplifying DNA in the fluid. The substantially monolithic structure also has defined therein a means for heating the DNA amplification chamber and a means for cooling the DNA amplification chamber.
In a second principal aspect, the present invention provides a DNA analysis system comprising a sample inlet port, a cell lysis chamber in fluid communication with the sample inlet port, a DNA separation chamber in fluid communication with said cell lysis chamber, a DNA amplification chamber in fluid communication with the DNA separation chamber, and a DNA detection system in fluid communication with the DNA amplification system. The DNA amplification chamber is defined by substantially monolithic structure that is formed from a plurality of green-sheet layers sintered together. The green-sheet layers contain particles selected from the group consisting of ceramic particles, glass particles, and glass-ceramic particles.
In a third principal aspect, the present invention provides a method for performing DNA analysis. A fluidic sample containing cells is placed in a cell lysis chamber. The cells in the cell lysis chamber are lysed to release cell contents, including sample DNA. The cell contents are passed to a DNA separation chamber. In the DNA separation chamber, the sample DNA is adsorbed onto a plurality of micro-beads and then eluted from the micro-beads. The sample DNA is passed to a DNA amplification chamber, where the sample DNA is amplified to produce amplified DNA. The amplified DNA is then detected. The cell lysis chamber, DNA separation chamber, and DNA amplification chamber are part of a fluid passageway defined in a substantially monolithic structure formed from a plurality of green-sheet layers sintered together. The green-sheet layers include particles selected from the group consisting of ceramic particles, glass particles, and glass-ceramic particles.